- Email: [email protected]
Incorporation of 4-thiouridine into RNA in germinating radish seeds
Volume 119, number 1
INCORPORATION
FEBS LETTERS
OF 4-THIOURIDINE
INTO RNA IN GERMINATING
September 1980
RADISH
SEEDS
Hitoshi SHIBATA, Hideo OCHIAI and Isamu UCHIDA Laboratory of Biochemistry, Colle
1. Introduction Animal cells metabolize sulfur-containing analogues of nucleic acid precursors into RNA. Rat liver slices [ 1 ] and mouse myeloma cells [2] incorporate 2-thiouridine into RNA. BHK-2 1/C cells (baby hamster kidney cell line) [3] can incorporate 6-thioguanosine and 4thiouridine into RNA. The relative incorporation of these sulfur-containing nucleosides into specific RNA species such as mRNA and rRNA appeared constant [2,3]. RNA containing sulfur-nucleosides can be separated from non-sulfur-containing RNA by affinity chromatography with mercurated-supports [2,3]. 4-Thiouridine occurs naturally as a minor constituent in bacterial tRNA [4-61, but not in plant tRNA [7]. The 4-thiouridine absorbed during the germination of seeds has an inhibitory effect on chloroplast development [&lo]. However, the metabolism of 4thiouridine in germinating seeds remains obscure. We now report the incorporation of 4-thiouridine into the RNA of germinating radish seeds and the separation of the 4-thiouridine-containing RNA by affinity chromatography on mercurated cellulose.
2. Materials and methods 4-Thiouridine was synthesized and purified as in [8]. The concentration of 4-thiouridine was determined using an absorbance coefficient of 21.5 X lo3 M-l . cm-’ at 331 nm [ll]. [‘4C]Methylamine-HC1 (50.1 mCi/mmol) was obtained from New England Nuclear. Oligo(dT)-cellulose was a product of Boehringer . Mercurated cellulose (Hg-cellulose) was prepared by the method in [12]. 2.1. Germination with 4-thiouridine
and grown with or without 0.5 mM 4-thiouridine in the dark for 4 days at 22-25”C, as detailed in [9]. 2.2. RNA preparation
Excised radish cotyledons were homogenized in an ice-cold mortar with 0.05 M Tris-HCl (pH 9.0) contaming 5 mM MgClz, 1% (w/v) sodium dodecyl sulfate, 0.2% (v/v) Triton X-l 00 and 5 pg/ml of polyvinyl sulfate (potassium salt). The homogenate was stirred vigorously for 30 min with an equal volume of phenolchloroform [phenol (adjusted to pH 9.0 with Tris): chloroform = 1: 1]. The aqueous phase was re-extracted with phenol-chloroform. RNA was precipitated from the aqueous phase by addition of 2 vol. ethanol and stored at -20°C. The precipitate was collected, washed with 80% ethanol, then dissolved in and dialyzed against water. The concentration of RNA was estimated assuming that 1 mg RNA/ml has an A&“mnm of 20 [ 131. The fractionation of RNA into poly(A)-RNA, high Mr RNA and low M, RNA was performed essentially by the methods in [3]. The RNA preparation dissolved in 10 mM Tris-HCl (pH 7.4) containing 0.5 M NaCl was applied to a column of oligo(dT)cellulose equilibrated with 0.5 M NaCl in 10 mM Tris-HCl (pH 7.4). After washing the column with the same buffer to elute poly(A)free RNA, the poly(A)containing RNA was eluted with 10 mM Tris-HCl (pH 7.4). Poly(A)-free RNA was precipitated with 2 vol. ethanol and after which it was dissolved in 3 ml 10 mM Tris-HCl (pH 7.4) containing 10 mM NaCl and 10 mM EDTA. LiCl(2 ml, 5 M) was added and the mixture was stored overnight at 4°C before centrifugation at 10 000 X g for 10 min. The pelleted, high Mr RNA was dissolved in and dialyzed against water. The supernatant containing low Mr RNA was dialyzed against water.
Radish seeds (Raphanus satimrs) were germinated ElsevierlNorth-Holland Biomedical Press
85
Volume
119, number
2.3. Derivertizatiotl
1
of RNA
4-Thiouridine-containing
0.6
RNA
(1.4
mg) was
2.4. Mercurated cellulose chromatography RNA in 4 mM Tris-HCl (pH 7.4) containing 0.5 M KC1 and 4 mM EDTA was applied to an Hg-cellulose column (0.9 X 5 cm) which had been equilibrated with the application buffer, monitoring the unbound RNA by Aza. The bound RNA was eluted with 50 mM 2-mercaptoethanol in 4 mM Tris-HCl (pH 7.4), monitoring the Asm. Under these conditions, at least, 100 pmol4-thiouridine bound to 1 g Hg-cellulose (0.9 X 5 cm) and was eluted with 2-mercaptoethanol; a recovery of 80-9070.
3. Results 3 .l . Incorporation of 4-thiouridine during germination
1980
r
incu-
bated with 7.8 mM [‘4C]methylamine (1 PCi, pH 10.4) and 7.8 mM NaI04, which was added last to initiate the reaction [l 11, in a 1 ml total vol. at 40°C for 90 min. At specified intervals, samples (100 ~1) were withdrawn and poured into 1.5 ml ice-cold 10% (w/v) trichloroacetic acid. The precipitate was collected on a Millipore paper and washed with 25 ml 10% trichloroacetic acid. Radioactivity incorporated into the RNA was measured in an aqueous scintillator system using an Aloka LSC-602 liquid scintillation counter.
into radish RNA
Radish seeds were germinated with 0.5 mM 4thiouridine containing 1 mM potassium phosphate (,pH 7.0) and were grown for 4 days in the dark. The concentrated RNA prepared from the 4-thiouridinecultured radish cotyledons showed a distinct peak at 331 nm that was 0.6-0.7% of the peak at 260 nm (fig.lA). The RNA preparation from control plants germinated and grown without 4-thiouridine lacked this peak (fig. 1B). Both diluted RNA preparations had absorption profiles characteristic of RNA, with an absorption minimum and a maximum at 232 and 258 nm, respectively (fig.lC). Since 4-thiouridine and 4-thiouridylate have maximum absorptions at 331 nm [4-61. as compared to the usual maximum near 260 nm, the presence of 4-thiouridine residues in the RNA can be monitored by this characteristic absorption maximum. 4-Thiouridine in Escherichia coli tRNAs reacts with hydrogen peroxide [5,6] or hydroxylamine [14] to give uridine ofp-hydroxycytidine, respectively. In addition, the 86
September
FEBS LETTERS
00
250
300
350
400
Fig.1. Absorption spectra of the RNA from radish cotyledons. Radish seeds were germinated and grown with 1 mM potassium phosphate (pH 7.0) with or without 0.5 mM 4thiourldine, in the dark at 22- 25°C for 4 days. The RNA isolated from 2 g excised cotyledons was dissolved in 5 ml water. (A) RNA from 0.5 mM 4-thiouridme-cultured radish cotyledons. (B) RNA from control cotyledons germinated and grown without 4-thiouridine. (C) The two RNA solutions diluted loo-fold with water.
4-thiouridine residues of tRNA are transformed in the presence of periodate and [‘4C]methylamine (at pH I 0.4) to labeled fl-methylcytidine residues [ 111. These chemical modifications occur selectively at the 4-thiouridine residues in RNA [5,6,11,14]. The effect of selective chemical modification by hydrogen peroxide (pt A) and hydroxylamine (pt B) on the absorption profile between 300 and 400 nm of the RNA preparation from the 4-thiouridine-cultured radish cotyledons is shown in fig. 2. The characteristic peak at 331 nm of the RNA disappeared with time due to selective chemical modification, evidence that 4-thiouridine is present in the RNA preparation. A residual absorption at 331 nm after the modification was attributed to the tail from the UV absorption of the concentrated RNA. because the control RNA containing no 4-thiouridine residues showed a similar absorption curve (fig. 1B). Thus. the contents of 4-thiouridine in RNA can be determined spectrophotometrically by the difference in A331 before and after the chemical modification of RNA with hydrogen peroxide. By this spectrophotometric analysis, we estimated that the RNA from 0.5 mM 4-thiouridine-cultured radish cotyledons contains 7-10 nmol 4-thiouridine/mg RNA (table 1). More-
Volume
119, number
1
FEBS LETTERS
September
\ o.4-l
1980
with NaI04
0
0
0
T 0
NaIOq
without
0
l
0
1. 0
60
30
90
Oi 300
Reaction
Fig.2. Effect of selective chemical modification on the absorption spectra of the RNA from 4-thioundine-cultured radish cotyledons: (A) treated with 0.176 M hydrogen peroxide in 10 mM Tris-HCl (pH 8.0), (B) treated with 0.5 M hydroxylamine in 10 mM potassmm phosphate (pH 7.0).
over, incubation of the RNA from 4-thiouridine-cultured radish cotyledons in [14C]methylamine [I l] resulted in the periodate-dependent incorporation of 14C from [‘4C]methylamine into the trichloroacetic acid-insoluble fraction (fig.3). These results also demonstrate the presence of 4-thiouridine in the RNA.
Properties
4-Thioundine (nmol/mg
Table 1 of 4-thiouridine-containing
in RNA RNA)C
Hg-cellulose-bound
RNA
Total RNAa
Chloroplast
1.28
3.15
3.94 5.72 2.2
3.60 3.38 1.3
RNAb
RNA
(% total amount) (A331 l.4260 X 100) (mol% 4-thiouridine)
d
a RNA prepared from 0.5 mM 4-thlouridinc-cultured, 4-dayold etiolated radish cotyledons b 4-Thiouridine-cultured, 4-day-old iadish seedlings were illuminated for 24 h (2500 lux). RNA was prepared from chloroplasts, which were isolated from greening radish cotyledons and purified by sucrose density gradient centrlfugation as in I91 ’ Determined spectrophotometrically as in the text d Calculated from the A,,, /A,,, X 100 ratio using the equation in II51
time
(min)
Fig.3. Kinetics of the incorporation of 14C from [14C]methylamine into RNA from 4-thiouridine-cultured radish cotyledons. RNA (1.4 mg) contaming 12.1 nmol4-thlouridine residues (determined spectrophotometrically as in the text) was incubated with L”Clmethylamme \vith or without NaIO, as in section 2. The assay was for trichloroacetic acid-msoluble radioactivity.
The incorporation of 14C reached a plateu when 8.6 nmol l4 C/mg RNA was bound. This value was m fair agreement with that obtained by the spectrophotometric method above. 3.2. Separation of the 4-thiouridine-containing RNA by affinity chromatography on mercurated cellulose The radish RNA from 0.5 mM 4-thiouridine-cultured etiolated cotyledons was applied to Hg-cellulose (fig.4). After washing out the unbound RNA, the bound RNA was eluted with Z-mercaptoethanol. Some 3-470 of the RNA bound to the mercurated cellulose. After dialysis to remove 2-mercaptoethanol, the bound RNA gave an Asa1 /A,,, X 100 ratio of 5.72. This value corresponds to 2.2 mol% of 4-thiouridine in the bound RNA (table 1). assuming that the Ass1 / A 26o X 100 ratio of 2.03 for E. coli tRNAs equals 0.875 mol% of the 4-thiouridine residue [ 1.51. The properties of chloroplast RNA from isolatedchloroplasts of 4-thiouridine-cultured radish cotyledons illuminated at 2500 lux for 34 h are shown in table 1. Chloroplast RNA also contained 4-thiouridine residues of -4 nmol/mg RNA. Some of the RNA bound to the Hg-cellulose and was eluted with mer87
Volume
119, number
FEBS LETTERS
1
September
1980
4. Discussion
Fig.4. Affinity chromatography of RNA on mercurated cellulose. RNA prepared from 0.5 rnM 4-thlourldine-cultured radish cotyledons was applied to an Hg-cellulose column (0.9 X 5 cm) equilibrated with 0.5 M KC1 containing 4 mM Tris-HCl (pH 7.4) and 4 mM EDTA. Non-bound RNA was eluted with KC1 solution. Bound RNA was eluted with 50 mM 2-mercaptoethanol in 4 mM Tris-HCl (pH 7.4). Elution of the bound RNA was monitored at A 330.
captoethanol. The mol% of 4-thiouridine in the bound chloroplast RNA was estimated to be 1.3. The total RNA preparation from 0.5 mM 4-thiouridine-cultured, etiolated radish cotyledons was fractionated into poly(A)-, high I@,, and low M, RNAs by the methods in [3]. The low M, and high M, RNAs showed distinct peaks at 331 nm, and their 4-thiouridine residues were spectrophotometricaly calculated to be 19.7 and 6.5 nmol/mg RNA, respectively (table 2). A part of each fractionated RNA bound to the Hg-cellulose, and was eluted with 2-mercaptoethanol, indicating the presence of 4-thiouridine in each RNA species (table 2). Thus, we concluded that the 4thiouridine absorbed by radish seeds is incorporated into mRNA, rRNA, and into chloroplast RNA.
Table 2 Properties of fractionated RNA species from 0.5 mM 4-thiouridine-cultured radish cotyledons RNA species
4-thiouridine (nmol/mg RNA)a
Hg-cellulose-bound (% total amount)
6.50 19.7
12.3 1.95 6.51
POSY (A)
High Mr Low M, a Determined
88
spectrophotometrically
as in the text
At least 3 sulfur-containing analogues of nucleic acid precursors. 6-thioguanosine [3], 2-thiouridine [1,2] and 4-thiouridine [3], are incorporated into the RNA in animal cells. The incorporation of these analogues into RNA was demonstrated mainly by the retention of the RNA on a mercurial support. We demonstrated more clearly the incorporation of 4-thiouridine into the RNA of germinating radish seeds by 4 different methods. Three methods: (1j the absorption maximum of isolated RNA at 331 nm (fig.l), (2) the selective chemical modlfication of the RNA (fig.?), and (3) the incorporation of radiocarbon into the RNA from [ 14C]methylamine in the presence of periodate (fig.3), provided the conclusive proof of the presence of 4-thiouridine residues in bacterial tRNAs [4-l 11. The fourth method was the retention of the RNA on Hg-cellulose (fig.4). Poly(4-thiouridylic acid) can act as mRNA for the synthesis of polyphenylalanine [ 161. Following treatment of baby hamster kidney cells with 4-thiouridine. the newly synthesized RNA containing 4-thiouridine occupied >60% of the total RNA (calculated from the results in [3]). However, the incorporation of 4thiouridine into RNA does not result in the inhibition of protein synthesis, or in the production of abnormal proteins [3]. The tRNAs of E. coli, strain B synthesized in the presence of 5-fluorouracil have up to a 100% replacement of the uridine residues by 5-fluorouracil, but the incorporation of .5-fluorouracil into tRNA does not affect its ability to accept amino acids [ 171. From these results and from the relatively low content (3-S% of the total RNA) of 4-thioundinecontaining RNA (table I), the inhibition of chloroplast development by the 4-thiouridine culture [8-l 0] might not be fully accounted for by the presence of 4thiouridine-containing RNAs. However, we observed [8] a reduced RNA synthesis at an early stage of germination of the 4-thiouridine-cultured radish cotyledons. Thus, 4-thiouridine, or one of its anabolized derivatives suchas 4-thiouridine-5’-triphosphate,probably inhibits RNA synthesis thereby causing the inhibition of chloroplast development.
Acknowledgement We thank Dr Kozi Asada of Kyoto University for carefully reading the manuscript.
Volume
119, number
1
References Ill Yu, M. W., Sedlak, J. and Lindsay, R. H. (1973) Arch. Biochem. Biophys. 155, 111-119. I21 Ono, M. and Kawakami, M. (1977) J. Biochem. 81, 1247-1252. I31 Melvin, W. T., Mdne, H. B., Slater, A. A., Allen, H. J. and Keir, M. M. (1978) Eur. J. Biochem. 92, 373-379. I41 Lipsett, M. N. (1965) J. Biol. Chem. 240, 3975-3978. ISI Shugart, L. (1972) Arch. Biochem. Biophys. 148, 488-495. I61 Rao, Y. S. P. andCherayiI, J. D. (1974) Biochem. J. 143, 2855294. I71 Young, P. A. and Kaiser, I. I. (1979) Plant Physiol. 63, 511-517. ISI Ochiai, H., Shibata, H. and Suekane, T. (1971) Agric, Biol. Chem. 35, 1259-1266.
FEBS LETTERS
September
19 80
19 1 Shibata, H. and Ochiai, H. (1980) Plant CeII Physiol. 21, 311-320. I101 Shibata, H., Ochiai, H. and Shimizu, A. (1980) Plant Ceil Physiol. 21, 321-329. Ill1 Ziff, E.B. and Fresco, J. (1969) Biochemistry 8, 32423248. 1121 Shainoff, J. R. (1968) J. Immunol. 100, 187-193. I131 Hartley, M. R. and Ellis, R. J. (1973) Biochem. J. 134, 249-262. 1141 Iida. S., Chung. K. C. and Hayatsu, H. (1973) Biochim. Biophys. Acta 308, 1988204. I151 Frendewey, D. A. and Kaiser, I. I. (1979) Biochemistry 18, 3179-3185. I161 Fiser, I., Scheit, K. H. and Kuechler, E. (1977) Eur. J. Biochem. 74,447-456. I17 I Lowrie, R. J. and Bergquist, P. L. (1968) Biochemistry 7,1761-1770.
89
INCORPORATION
FEBS LETTERS
OF 4-THIOURIDINE
INTO RNA IN GERMINATING
September 1980
RADISH
SEEDS
Hitoshi SHIBATA, Hideo OCHIAI and Isamu UCHIDA Laboratory of Biochemistry, Colle
1. Introduction Animal cells metabolize sulfur-containing analogues of nucleic acid precursors into RNA. Rat liver slices [ 1 ] and mouse myeloma cells [2] incorporate 2-thiouridine into RNA. BHK-2 1/C cells (baby hamster kidney cell line) [3] can incorporate 6-thioguanosine and 4thiouridine into RNA. The relative incorporation of these sulfur-containing nucleosides into specific RNA species such as mRNA and rRNA appeared constant [2,3]. RNA containing sulfur-nucleosides can be separated from non-sulfur-containing RNA by affinity chromatography with mercurated-supports [2,3]. 4-Thiouridine occurs naturally as a minor constituent in bacterial tRNA [4-61, but not in plant tRNA [7]. The 4-thiouridine absorbed during the germination of seeds has an inhibitory effect on chloroplast development [&lo]. However, the metabolism of 4thiouridine in germinating seeds remains obscure. We now report the incorporation of 4-thiouridine into the RNA of germinating radish seeds and the separation of the 4-thiouridine-containing RNA by affinity chromatography on mercurated cellulose.
2. Materials and methods 4-Thiouridine was synthesized and purified as in [8]. The concentration of 4-thiouridine was determined using an absorbance coefficient of 21.5 X lo3 M-l . cm-’ at 331 nm [ll]. [‘4C]Methylamine-HC1 (50.1 mCi/mmol) was obtained from New England Nuclear. Oligo(dT)-cellulose was a product of Boehringer . Mercurated cellulose (Hg-cellulose) was prepared by the method in [12]. 2.1. Germination with 4-thiouridine
and grown with or without 0.5 mM 4-thiouridine in the dark for 4 days at 22-25”C, as detailed in [9]. 2.2. RNA preparation
Excised radish cotyledons were homogenized in an ice-cold mortar with 0.05 M Tris-HCl (pH 9.0) contaming 5 mM MgClz, 1% (w/v) sodium dodecyl sulfate, 0.2% (v/v) Triton X-l 00 and 5 pg/ml of polyvinyl sulfate (potassium salt). The homogenate was stirred vigorously for 30 min with an equal volume of phenolchloroform [phenol (adjusted to pH 9.0 with Tris): chloroform = 1: 1]. The aqueous phase was re-extracted with phenol-chloroform. RNA was precipitated from the aqueous phase by addition of 2 vol. ethanol and stored at -20°C. The precipitate was collected, washed with 80% ethanol, then dissolved in and dialyzed against water. The concentration of RNA was estimated assuming that 1 mg RNA/ml has an A&“mnm of 20 [ 131. The fractionation of RNA into poly(A)-RNA, high Mr RNA and low M, RNA was performed essentially by the methods in [3]. The RNA preparation dissolved in 10 mM Tris-HCl (pH 7.4) containing 0.5 M NaCl was applied to a column of oligo(dT)cellulose equilibrated with 0.5 M NaCl in 10 mM Tris-HCl (pH 7.4). After washing the column with the same buffer to elute poly(A)free RNA, the poly(A)containing RNA was eluted with 10 mM Tris-HCl (pH 7.4). Poly(A)-free RNA was precipitated with 2 vol. ethanol and after which it was dissolved in 3 ml 10 mM Tris-HCl (pH 7.4) containing 10 mM NaCl and 10 mM EDTA. LiCl(2 ml, 5 M) was added and the mixture was stored overnight at 4°C before centrifugation at 10 000 X g for 10 min. The pelleted, high Mr RNA was dissolved in and dialyzed against water. The supernatant containing low Mr RNA was dialyzed against water.
Radish seeds (Raphanus satimrs) were germinated ElsevierlNorth-Holland Biomedical Press
85
Volume
119, number
2.3. Derivertizatiotl
1
of RNA
4-Thiouridine-containing
0.6
RNA
(1.4
mg) was
2.4. Mercurated cellulose chromatography RNA in 4 mM Tris-HCl (pH 7.4) containing 0.5 M KC1 and 4 mM EDTA was applied to an Hg-cellulose column (0.9 X 5 cm) which had been equilibrated with the application buffer, monitoring the unbound RNA by Aza. The bound RNA was eluted with 50 mM 2-mercaptoethanol in 4 mM Tris-HCl (pH 7.4), monitoring the Asm. Under these conditions, at least, 100 pmol4-thiouridine bound to 1 g Hg-cellulose (0.9 X 5 cm) and was eluted with 2-mercaptoethanol; a recovery of 80-9070.
3. Results 3 .l . Incorporation of 4-thiouridine during germination
1980
r
incu-
bated with 7.8 mM [‘4C]methylamine (1 PCi, pH 10.4) and 7.8 mM NaI04, which was added last to initiate the reaction [l 11, in a 1 ml total vol. at 40°C for 90 min. At specified intervals, samples (100 ~1) were withdrawn and poured into 1.5 ml ice-cold 10% (w/v) trichloroacetic acid. The precipitate was collected on a Millipore paper and washed with 25 ml 10% trichloroacetic acid. Radioactivity incorporated into the RNA was measured in an aqueous scintillator system using an Aloka LSC-602 liquid scintillation counter.
into radish RNA
Radish seeds were germinated with 0.5 mM 4thiouridine containing 1 mM potassium phosphate (,pH 7.0) and were grown for 4 days in the dark. The concentrated RNA prepared from the 4-thiouridinecultured radish cotyledons showed a distinct peak at 331 nm that was 0.6-0.7% of the peak at 260 nm (fig.lA). The RNA preparation from control plants germinated and grown without 4-thiouridine lacked this peak (fig. 1B). Both diluted RNA preparations had absorption profiles characteristic of RNA, with an absorption minimum and a maximum at 232 and 258 nm, respectively (fig.lC). Since 4-thiouridine and 4-thiouridylate have maximum absorptions at 331 nm [4-61. as compared to the usual maximum near 260 nm, the presence of 4-thiouridine residues in the RNA can be monitored by this characteristic absorption maximum. 4-Thiouridine in Escherichia coli tRNAs reacts with hydrogen peroxide [5,6] or hydroxylamine [14] to give uridine ofp-hydroxycytidine, respectively. In addition, the 86
September
FEBS LETTERS
00
250
300
350
400
Fig.1. Absorption spectra of the RNA from radish cotyledons. Radish seeds were germinated and grown with 1 mM potassium phosphate (pH 7.0) with or without 0.5 mM 4thiourldine, in the dark at 22- 25°C for 4 days. The RNA isolated from 2 g excised cotyledons was dissolved in 5 ml water. (A) RNA from 0.5 mM 4-thiouridme-cultured radish cotyledons. (B) RNA from control cotyledons germinated and grown without 4-thiouridine. (C) The two RNA solutions diluted loo-fold with water.
4-thiouridine residues of tRNA are transformed in the presence of periodate and [‘4C]methylamine (at pH I 0.4) to labeled fl-methylcytidine residues [ 111. These chemical modifications occur selectively at the 4-thiouridine residues in RNA [5,6,11,14]. The effect of selective chemical modification by hydrogen peroxide (pt A) and hydroxylamine (pt B) on the absorption profile between 300 and 400 nm of the RNA preparation from the 4-thiouridine-cultured radish cotyledons is shown in fig. 2. The characteristic peak at 331 nm of the RNA disappeared with time due to selective chemical modification, evidence that 4-thiouridine is present in the RNA preparation. A residual absorption at 331 nm after the modification was attributed to the tail from the UV absorption of the concentrated RNA. because the control RNA containing no 4-thiouridine residues showed a similar absorption curve (fig. 1B). Thus. the contents of 4-thiouridine in RNA can be determined spectrophotometrically by the difference in A331 before and after the chemical modification of RNA with hydrogen peroxide. By this spectrophotometric analysis, we estimated that the RNA from 0.5 mM 4-thiouridine-cultured radish cotyledons contains 7-10 nmol 4-thiouridine/mg RNA (table 1). More-
Volume
119, number
1
FEBS LETTERS
September
\ o.4-l
1980
with NaI04
0
0
0
T 0
NaIOq
without
0
l
0
1. 0
60
30
90
Oi 300
Reaction
Fig.2. Effect of selective chemical modification on the absorption spectra of the RNA from 4-thioundine-cultured radish cotyledons: (A) treated with 0.176 M hydrogen peroxide in 10 mM Tris-HCl (pH 8.0), (B) treated with 0.5 M hydroxylamine in 10 mM potassmm phosphate (pH 7.0).
over, incubation of the RNA from 4-thiouridine-cultured radish cotyledons in [14C]methylamine [I l] resulted in the periodate-dependent incorporation of 14C from [‘4C]methylamine into the trichloroacetic acid-insoluble fraction (fig.3). These results also demonstrate the presence of 4-thiouridine in the RNA.
Properties
4-Thioundine (nmol/mg
Table 1 of 4-thiouridine-containing
in RNA RNA)C
Hg-cellulose-bound
RNA
Total RNAa
Chloroplast
1.28
3.15
3.94 5.72 2.2
3.60 3.38 1.3
RNAb
RNA
(% total amount) (A331 l.4260 X 100) (mol% 4-thiouridine)
d
a RNA prepared from 0.5 mM 4-thlouridinc-cultured, 4-dayold etiolated radish cotyledons b 4-Thiouridine-cultured, 4-day-old iadish seedlings were illuminated for 24 h (2500 lux). RNA was prepared from chloroplasts, which were isolated from greening radish cotyledons and purified by sucrose density gradient centrlfugation as in I91 ’ Determined spectrophotometrically as in the text d Calculated from the A,,, /A,,, X 100 ratio using the equation in II51
time
(min)
Fig.3. Kinetics of the incorporation of 14C from [14C]methylamine into RNA from 4-thiouridine-cultured radish cotyledons. RNA (1.4 mg) contaming 12.1 nmol4-thlouridine residues (determined spectrophotometrically as in the text) was incubated with L”Clmethylamme \vith or without NaIO, as in section 2. The assay was for trichloroacetic acid-msoluble radioactivity.
The incorporation of 14C reached a plateu when 8.6 nmol l4 C/mg RNA was bound. This value was m fair agreement with that obtained by the spectrophotometric method above. 3.2. Separation of the 4-thiouridine-containing RNA by affinity chromatography on mercurated cellulose The radish RNA from 0.5 mM 4-thiouridine-cultured etiolated cotyledons was applied to Hg-cellulose (fig.4). After washing out the unbound RNA, the bound RNA was eluted with Z-mercaptoethanol. Some 3-470 of the RNA bound to the mercurated cellulose. After dialysis to remove 2-mercaptoethanol, the bound RNA gave an Asa1 /A,,, X 100 ratio of 5.72. This value corresponds to 2.2 mol% of 4-thiouridine in the bound RNA (table 1). assuming that the Ass1 / A 26o X 100 ratio of 2.03 for E. coli tRNAs equals 0.875 mol% of the 4-thiouridine residue [ 1.51. The properties of chloroplast RNA from isolatedchloroplasts of 4-thiouridine-cultured radish cotyledons illuminated at 2500 lux for 34 h are shown in table 1. Chloroplast RNA also contained 4-thiouridine residues of -4 nmol/mg RNA. Some of the RNA bound to the Hg-cellulose and was eluted with mer87
Volume
119, number
FEBS LETTERS
1
September
1980
4. Discussion
Fig.4. Affinity chromatography of RNA on mercurated cellulose. RNA prepared from 0.5 rnM 4-thlourldine-cultured radish cotyledons was applied to an Hg-cellulose column (0.9 X 5 cm) equilibrated with 0.5 M KC1 containing 4 mM Tris-HCl (pH 7.4) and 4 mM EDTA. Non-bound RNA was eluted with KC1 solution. Bound RNA was eluted with 50 mM 2-mercaptoethanol in 4 mM Tris-HCl (pH 7.4). Elution of the bound RNA was monitored at A 330.
captoethanol. The mol% of 4-thiouridine in the bound chloroplast RNA was estimated to be 1.3. The total RNA preparation from 0.5 mM 4-thiouridine-cultured, etiolated radish cotyledons was fractionated into poly(A)-, high I@,, and low M, RNAs by the methods in [3]. The low M, and high M, RNAs showed distinct peaks at 331 nm, and their 4-thiouridine residues were spectrophotometricaly calculated to be 19.7 and 6.5 nmol/mg RNA, respectively (table 2). A part of each fractionated RNA bound to the Hg-cellulose, and was eluted with 2-mercaptoethanol, indicating the presence of 4-thiouridine in each RNA species (table 2). Thus, we concluded that the 4thiouridine absorbed by radish seeds is incorporated into mRNA, rRNA, and into chloroplast RNA.
Table 2 Properties of fractionated RNA species from 0.5 mM 4-thiouridine-cultured radish cotyledons RNA species
4-thiouridine (nmol/mg RNA)a
Hg-cellulose-bound (% total amount)
6.50 19.7
12.3 1.95 6.51
POSY (A)
High Mr Low M, a Determined
88
spectrophotometrically
as in the text
At least 3 sulfur-containing analogues of nucleic acid precursors. 6-thioguanosine [3], 2-thiouridine [1,2] and 4-thiouridine [3], are incorporated into the RNA in animal cells. The incorporation of these analogues into RNA was demonstrated mainly by the retention of the RNA on a mercurial support. We demonstrated more clearly the incorporation of 4-thiouridine into the RNA of germinating radish seeds by 4 different methods. Three methods: (1j the absorption maximum of isolated RNA at 331 nm (fig.l), (2) the selective chemical modlfication of the RNA (fig.?), and (3) the incorporation of radiocarbon into the RNA from [ 14C]methylamine in the presence of periodate (fig.3), provided the conclusive proof of the presence of 4-thiouridine residues in bacterial tRNAs [4-l 11. The fourth method was the retention of the RNA on Hg-cellulose (fig.4). Poly(4-thiouridylic acid) can act as mRNA for the synthesis of polyphenylalanine [ 161. Following treatment of baby hamster kidney cells with 4-thiouridine. the newly synthesized RNA containing 4-thiouridine occupied >60% of the total RNA (calculated from the results in [3]). However, the incorporation of 4thiouridine into RNA does not result in the inhibition of protein synthesis, or in the production of abnormal proteins [3]. The tRNAs of E. coli, strain B synthesized in the presence of 5-fluorouracil have up to a 100% replacement of the uridine residues by 5-fluorouracil, but the incorporation of .5-fluorouracil into tRNA does not affect its ability to accept amino acids [ 171. From these results and from the relatively low content (3-S% of the total RNA) of 4-thioundinecontaining RNA (table I), the inhibition of chloroplast development by the 4-thiouridine culture [8-l 0] might not be fully accounted for by the presence of 4thiouridine-containing RNAs. However, we observed [8] a reduced RNA synthesis at an early stage of germination of the 4-thiouridine-cultured radish cotyledons. Thus, 4-thiouridine, or one of its anabolized derivatives suchas 4-thiouridine-5’-triphosphate,probably inhibits RNA synthesis thereby causing the inhibition of chloroplast development.
Acknowledgement We thank Dr Kozi Asada of Kyoto University for carefully reading the manuscript.
Volume
119, number
1
References Ill Yu, M. W., Sedlak, J. and Lindsay, R. H. (1973) Arch. Biochem. Biophys. 155, 111-119. I21 Ono, M. and Kawakami, M. (1977) J. Biochem. 81, 1247-1252. I31 Melvin, W. T., Mdne, H. B., Slater, A. A., Allen, H. J. and Keir, M. M. (1978) Eur. J. Biochem. 92, 373-379. I41 Lipsett, M. N. (1965) J. Biol. Chem. 240, 3975-3978. ISI Shugart, L. (1972) Arch. Biochem. Biophys. 148, 488-495. I61 Rao, Y. S. P. andCherayiI, J. D. (1974) Biochem. J. 143, 2855294. I71 Young, P. A. and Kaiser, I. I. (1979) Plant Physiol. 63, 511-517. ISI Ochiai, H., Shibata, H. and Suekane, T. (1971) Agric, Biol. Chem. 35, 1259-1266.
FEBS LETTERS
September
19 80
19 1 Shibata, H. and Ochiai, H. (1980) Plant CeII Physiol. 21, 311-320. I101 Shibata, H., Ochiai, H. and Shimizu, A. (1980) Plant Ceil Physiol. 21, 321-329. Ill1 Ziff, E.B. and Fresco, J. (1969) Biochemistry 8, 32423248. 1121 Shainoff, J. R. (1968) J. Immunol. 100, 187-193. I131 Hartley, M. R. and Ellis, R. J. (1973) Biochem. J. 134, 249-262. 1141 Iida. S., Chung. K. C. and Hayatsu, H. (1973) Biochim. Biophys. Acta 308, 1988204. I151 Frendewey, D. A. and Kaiser, I. I. (1979) Biochemistry 18, 3179-3185. I161 Fiser, I., Scheit, K. H. and Kuechler, E. (1977) Eur. J. Biochem. 74,447-456. I17 I Lowrie, R. J. and Bergquist, P. L. (1968) Biochemistry 7,1761-1770.
89